(1) Field of the Invention
The present invention relates to a process for purifying insulin and insulin analogs that comprises high-pressure liquid chromatography with an acidic cation exchange medium performed in the presence of a water miscible organic modifier and at an elevated temperature followed by reverse phase chromatography performed in the presence of a water miscible organic modifier and at an elevated temperature.
(2) Description of Related Art
Recombinant production of insulin and insulin analogs in genetically modified microbial or yeast host cells entails expression of the insulin or insulin analog in the host cell as a single-chain precursor insulin molecule comprising three polypeptide domains (A-chain, C-chain, B-chain), as in native pro-insulins, along with the addition of a fusion peptide at the N-terminus whose function is to protect the nascent protein from internal degradation/modification during synthesis in the bacterial host, improve protein expression in the host, and to add a trypsin cleavage site that will render the correct amino acid, phenylalanine, at position B 1, post-digest
In the microbial host E. coli, the single-chain precursor insulin molecule is sequestered in inclusion bodies consisting of mostly of incorrectly folded single-chain precursor molecules. To produce recombinant insulin, the inclusion bodies are extracted from the cell, washed, and the single-chain precursor molecule is solubilized, refolded, and then purified by at least one chromatographic step. The refolded, single-chain precursor molecule is then further processed into insulin by the concurrent removal of the C-chain and the N-terminal fusion peptide by enzymatic digestion. Insulin is comprised of an A-chain and B-chain linked together by three disulfide bridges. In subsequent purification steps, including ion-exchange chromatography and reverse phase HPLC, insulin is purified away from digestion byproducts to yield a highly purified product. The purified product may be formulated in a zinc and m-cresol (preservative) containing buffer to provide the insulin drug product.
Methods for isolating inclusion bodies, refolding and enzymatically digesting precursor insulin molecules to produce insulin have been disclosed U.S. Pat. Nos. 5,663,291; 5,986,048; 6,380,355, and 5,473,049. Ion-exchange chromatography methods for purifying insulin from digestion byproducts have been disclosed in U.S. Pat. No. 5,101,013, which discloses cation-exchange chromatography on strongly acidic ion exchangers under atmospheric or medium pressure and elution by means of aqueous alkanol with only a relatively small amount of alkanol to purify insulin; and, U.S. Pat. No. 5,977,297, which discloses high-pressure cation-exchange chromatography on pressure-stable acidic cation exchangers under a pressure of about 1.1 MPa (11 bar) to about 40 MPa (400 bar) to purify insulin. Further purification of insulin has been described in U.S. Pat. Nos. 6,710,167 and 5,621,073.
While there are methods available for purifying insulin, there remains a need for alternative methods for purifying insulin and insulin analogs thereof.